Ecdysone 20-hydroxylation in Manduca sexta midgut: Kinetic parameters of mitochondrial and microsomal ecdysone 20-monooxygenases

The larval midgut of the tobacco hornworm, Manduca sexta, has high ecdysone 20-monooxygenase (E20MO) activity, located both in the mitochondria and in the microsomes. The apparent kinetic parameters for E20MO in mitochondria and microsomes were determined. The K_m⁵ (for ecdysone) of the mitochondrial and microsomal enzymes were 1.63 × 10⁻⁵ and 3.67 × 10⁻⁷ M, respectively. The V_max was 82.7 pmol/min/mg protein for mitochondria and 32.0 pmol/min/mg protein for microsomes. Although the mitochondrial E20MO has the higher V_max, at physiological ecdysone concentrations (10⁻⁷ − 10⁻⁸ M) it is only one-eighth to one-tenth as active as the microsomal enzyme. It is concluded that the microsomal E20MO is the primary, if not the only, enzyme involved in ecdysone 20-hydroxylation in M. sexta midgut. © 1996 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

Allelochemical stimulation of ecdysone 20-monooxygenase in fall armyworm larvae

The influence of dietary allelochemical on ecdysone 20-monooxygenase activity was studied in the fall armyworm, Spodoptera frugiperda (J.E. Smith). Feeding the indoles (indole-3-carbinol, indole-3-acetonitrile), flavonoids (flavone, β-naphthoflavone), monoterpenes (menthol, menthone, peppermint oil), and a coumarin (xanthotoxin) to the larvae stimulated midgut microsomal ecdysone 20-monooxygenase activity from 28 to 200% as compared with the controls. β-Naphthoflavone was the most potent inducer among those tested. Phenobarbital, a well-known cytochrome P450 inducer, also caused a 2-fold increase in the microsomal ecdysone 20-monooxygenase activity. Ecdysone 20-monooxygenase activity was 2.7-fold higher in the microsomal fraction than in the mitochondrial fraction isolated from larval midguts. Microsomal ecdysone 20-monooxygenase activity was highest in the fat body, followed by the midgut and Malpighian tubules. Tissue localization and enzyme inducibility were different between ecdysone 20-monooxygenase and xenobiotic-metabolizing cytochrome P450 monooxygenases, including aldrin epoxidase, biphenyl hydroxylase, methoxyresorufin O-demethylase, 7-ethoxycoumarin O-deethylase, p-chloro-N-methylaniline N-demethylase, and phorate sulfoxidase in fall armyworm larvae. © 1995 Wiley-Liss, Inc.     

The roles of cytochrome b5 in a reconstituted N-demethylase system containing cytochrome P-450.

Compound 102804 isolated from $\text{Bacillus cereus}$ has been found to be a potent inhibitor of the N⁵-methyltetrahydrofolate-homocysteine transmethylase isolated from $\text{Escherichia coli}$ B. This inhibition was noted when 102804 was added to the enzyme reaction mixture after the reaction started or concurrently with the preparation of the mixture. Chemically inactivated 102804 has no activity as an inhibitor of this enzyme system.     

Purification and some properties of cytochrome P-450 specific for steroid 17 alpha-hydroxylation and C17-C20 bond cleavage from guinea pig adrenal microsomes

The energy requirements for mitochondrial protein synthesis were investigated in isolated rat liver mitochondria. Controlled changes in coupling efficiency were obtained by titration with FCCP in the presence of various substrates. No relationship was observed between the efficiency of oxidative phosphorylation and the inhibition of protein synthesis. With succinate-ADP as the substrate the ADP:O ratio was decreased by 70–80% with no effect on protein synthesis. In contrast, with acetate-ADP as substrate, a 10–20% reduction in the ADP:O ratio gave complete inhibition of protein synthesis. The data suggest that the rate of ATP production is more important for maintenance of protein synthesis than the efficiency of coupling $\text{per se}$. Thus, certain substrates can support maximal rates of protein synthesis even in relatively poorly coupled mitochondria. Analysis of mitochondrial translation products formed in the presence of increasing FCCP concentrations also showed that decreased efficiency of oxidative phosphorylation had no influence on the nature of the products.     

New players in the regulation of ecdysone biosynthesis

Insect ecdysone steroid hormone regulates major developmental transitions, such as molting and metamorphosis. The production of ecdysone correlates well with the timing of these transitions. Finding out how the ecdysone biosynthesis is regulated is crucial to fully understand these sophisticated developmental switches. Here we summarized recent findings in the regulation of ecdysone biosynthesis from the aspects of cell signaling, key biosynthetic enzymes and substrate cholesterol trafficking.     

The obligatory requirement of cytochrome b5 in the p-nitroanisole O-demethylation reaction catalyzed by cytochrome P-450 with a high affinity for cytochrome b5

The role of cytochrome $\text{b}$₅ in the $\text{p}$-nitroanisole O-demethylation was studied with a reconstituted system containing a unique cytochrome P-450, isolated from rabbit liver microsomes as a species with a high affinity for cytochrome $\text{b}$₅. The maximal activity was obtained in the complete system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, NADH-cytochrome $\text{b}$₅ reductase, and Triton X-100 in addition to cytochrome $\text{b}$₅. The omission of cytochrome $\text{b}$₅ from the complete system entirely abolished the activity. These results clearly show that cytochrome $\text{b}$₅ is obligatory in the reconstitute p-nitroanisole O-demethylation system, and this cytochrome P-450 probably interacts with cytochrome $\text{b}$₅ in such a way that the second electron is transferred from cytochrome $\text{b}$₅ and thus exhibits the demethylase activity.     

Effects of the compounds 2-methoxynaphthoquinone, 2-propoxynaphthoquinone, and 2-isopropoxynaphthoquinone on ecdysone 20-monooxygenase activity

The effects of the natural compound 2-methoxy-1,4-naphthoquinone, isolated from the leaves of Impatiens glandulifera and the synthetic compounds 2-propoxy-1,4-naphthoquinone and 2-isopropoxy-1,4-naphthoquinone on ecdysone 20-monooxygenase (E-20-M) activity were examined in three insect species. Homogenates of wandering stage third instar larvae of Drosophila melanogaster, or abdomens from adult female Aedes aegypti, or fat body or midgut from fifth instar larvae of Manduca sexta were incubated with radiolabelled ecdysone and increasing concentrations (from 1 x 10(-8) to 1 x 10(-3) M) of the three compounds. All three compounds were found to inhibit in a dose-dependent fashion the E-20-M activity in the three insect species. The concentration of these compounds required to elicit a 50% inhibition of this steroid hydroxylase activity in the three insect species examined ranged from approximately 3 x 10(-5) to 7 x 10(-4) M.     

Phytochrome-mediated regulation of a monooxygenase hydroxylating cinnamic acid in etiolated pea seedlings

Cinnamic acid is hydroxylated by the mixed-function oxidase trans-cinnamic acid 4-hydroxylase (CA4H). The hydroxylation reaction involves the transfer of electrons from reduced pyridine nucleotides via the enzyme NADPH cytochrome P-450 reductase to the terminal oxidase cytochrome P-450. This multi-enzyme complex is localized in the microsomal fraction. Isopycnic and velocity gradient centrifugation suggest that in the apical bud of etiolated pea seedlings this complex is restricted to the endoplasmic reticulum membranes. CA4H activity which develops in dark germinating pea seedlings was found to be stimulated by light, an effect mediated by phytochrome. CA4H and NADPH cytochrome c reductase activities, cytochromes P-450 and b 5 contents were measured in seedlings submitted to either short pulses of red and far-red light, or to continuous far-red or blue irradiation. The results are discussed in terms of a specific effect of phytochrome on the different parts of the multi-enzyme complex.     

Cytochrome P-450-dependent catabolism of triethanolamine in Rhodotorula mucilaginosa

The yeast Rhodotorula mucilaginosa was able to grow in media containing triethanolamine or diethanolamine as the sole nitrogen source. During growth in the presence of triethanolamine, extracts of yeast cells contained increased levels of cytochrome P-450 dependent monooxygenase which catalyzed the oxidative N-dealkylation of aminoalcohols. Formation of diethanolamine, ethanolamine and glyoxylate from triethanolamine was demonstrated, and the identity of the products was verified by thin layer chromatography. These observations suggested the following scheme of triethanolamine catabolism: triethanolamine diethanolamine + glycolaldehyde, diethanolamine ethanolamine + glycolaldehyde, ethanolamine NH₃ + glycolaldehyde glycolate glyoxylate glycerate pathway.     

A mechanism for the loss of cytochrome P-450 in primary mouse hepatocytes

This study examined various biochemical parameters such as mitochondria and mitochondrial DNA (mtDNA), total heme and cyto P450 content in fresh hepatocytes and dedifferentiated hepatocytes. These parameters were chosen in order to understand the dramatic decrease in drug metabolism in cultured hepatocytes. The data in this study shows a temporal decrease in cytochrome P450, total heme and also a decrease in mitochondria. Also, the ratio of mtDNA content to mitochondrial density was found to increase as hepatocytes underwent dedifferentiation. Stereological analysis of cell preparations provided a measure of mitochondrial density per cell area and mtDNA content was assessed by the use of a specific radiolabelled probe. This study demonstrates that a loss of the organelle which is partially responsible for synthesis of heme correlates with a decrease in cytochrome P450.     

Decreased intracellular proteolysis correlates with the maintenance of a specific isoenzyme of cytochrome P-450

The rates of intracellular protein degradation, of identically labelled populations of proteins, were compared in hepatocytes cultured at 37 degrees (on an adsorbed collagen layer) and in cells preserved on gelatin gels at 10 degrees C. The half-lives of the long-lived proteins were 35.4+/-8.6 h (N=4) and 692.9+/-216.9 h (N=4) respectively. Proteolysis was substantially decreased at 10 degrees C but the rate of decrease remained constant. Hepatocytes rapidly removed resorufin from the culture medium. The resorufin was not being conjugated or accumulated within the cells. Dicumarol, a potent inhibitor of quinone oxidoreductase, at high concentration (500 microm ) caused only a 72% decrease in the utilization of resorufin. The microsomal detoxifying enzyme, cytochrome P-450 1A1 remained at a constant level in the preserved hepatocyte monolayers. The results of this study strongly favour storing hepatocytes at 10 degrees C rather than at 4 degrees or 37 degrees C.     

Ecdysone 20-hydroxylation in imaginal wing discs of Pieris brassicae (lepidoptera): Correlations with ecdysone and 20-hydroxyecdysone titers in pupae

Ecdysone 20-hydroxylase activity has been detected in pupal wing discs of Pieris brassicae. This activity is due to an enzyme system located in microsomal fractions. Its apparent Km is 58 nM for ecdysone. The enzyme is inhibited by the reaction product 20-hydroxyecdysone with an apparent Ki of 2.6 μM. Its activity varied during pupal-adult development with a maximum on day 4, when ecdysone levels are the highest in the animal. Although low, the peak activity is sufficient to assure 25% of the conversion of endogenous ecdysone into 20-hydroxyecdysone in pupae. Ecdysone and 20-hydroxyecdysone levels were measured in hemolymph and whole animals; ecdysone appears to be mainly located in hemolymph, whereas 20-hydroxyecdysone seems to be equally distributed between hemolymph and tissues. All these findings are discussed in relation to the roles of ecdysone and 20-hydroxyecdysone during pupal-adult development.     

The Induction of Cytochrome P-450 and Ethanol Oxidation in Yarrowia lipolytica Cells

The study of the effect of different ethanol concentrations in the medium on the growth and activity of enzymatic systems involved in ethanol oxidation in Yarrowia lipolytica showed that the cultivation of yeast cells on 1 and 2% ethanol caused their rapid growth and a drastic increase in cell respiration and sensitivity to cyanide already in the first hours of cultivation. At the same time, during cultivation on 3, 4, and 5% ethanol, the growth and respiration of yeast cells were considerably suppressed. All of the ethanol concentrations studied induced the synthesis of cytochrome P-450, its dynamics in cells being dependent on the initial concentration of ethanol in the medium. When the initial concentration of ethanol was 1 and 2%, the content of cytochrome P-450 in cells steeply decreased after a short period of induction. However, when the initial concentration of ethanol in the medium was 4 to 5%, the content of cytochrome P-450 in cells was high throughout the cultivation period. The induction of cytochrome P-450 in cells preceded the induction of the NAD-dependent enzymes alcohol dehydrogenase and catalase, which, like cytochrome P-450, are also involved in ethanol oxidation by yeasts. The activity of catalase was higher in the yeast cells grown in the presence of 3 to 5% ethanol than in the cells grown in the presence of 1 and 2% ethanol. The roles played by cytochrome P-450, alcohol dehydrogenase, and catalase in ethanol oxidation by yeast cells are discussed.     

Induction of cytochrome P-450-dependent drug metabolism by juvenile hormone mimics in mammalian cell culture

The monooxygenase activity of fetal hepatocytes in culture shows a differential response toward juvenile hormone I and analogs. Juvenile hormone I, R-20458, and Methoprene increase the deethyiation of 7-ethoxyresorufin while not affecting or even inhibiting the N-demethylation of p-chloro-N-methylaniline. RO-203600, a 1,3-benzodioxole-containing analog, increases both the deethylase and the N-demethylase, whereas Hydroprene does not affect either activity. The inductive effect with juvenile hormone I is obtained with exposure periods of at least 30 min and is maximum when the concentration of the hormone is 14 μM in the medium. This amount results in the covalent binding to cellular macromolecules of 1.3 × 19^?18 moles/cell. The induction requires continuous protein synthesis but RNA synthesis only for a short initial period. It is concluded that juvenile hormone and mimics induce specific cytochrome P-450 species in fetal liver cells even if the culture conditions are not optimal. The toxicological implications of these results are briefly discussed.     

Induction of cytochrome P-450 isozymes by hexachlorobenzene in rats and aromatic hydrocarbon (Ah)—Responsive mice

Hexachlorobenzene (HCB) differs markedly from other chlorinated benzenes (CBs) as an inducer of cytochrome P-450 (P-450) isozymes as determined by radioimmunoassay and immunoblotting. At > 99% pure, HCB induced both the phenobarbital-inducible forms, cytochromes P-450b + e (70X), and the 3-methylcholanthrene-inducible forms, cytochromes P-450c (58X) and P-450d (8X), in rat liver microsomes. The concentration of P-450d was considerably greater than that of P-450c in HCB-induced rat liver. In contrast to HCB, all lower chlorinated benzenes tested were PB-type inducers. Hexachlorobenzene increased the amounts of translatable messenger RNAs (mRNAs) for P-450b, P-450c, and P-450d in rat liver polysomes, suggesting that it increases the synthesis of these proteins. Evidence that HCB interacted with the putative Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was equivocal. Western blots of liver microsomes from Ahresponsive C57BL/6J (B6) and nonresponsive DBA/2J (D2) mice demonstrated that HCB produced a large increase in P₃-450 and a very small increase in P₁-450 in the responsive strain. The increase in P₁-450 was not observed after HCB administration to nonresponsive mice, but a small increase in P₃-450 was noted. These findings suggested that HCB may act through the Ah receptor. However, HCB was at best a very weak competitor for specific binding of [³H]-TCDD to the putative receptor in rat or mouse hepatic cytosol in vitro, producing decreases in binding of [³H]-TCDD only at very high concentrations (10^?6 to 10^?5 M).     

Differential expression of CYP6A5 and CYP6A5v2 in pyrethroid-resistant house flies, Musca domestica

Two cytochrome P450 alleles, CYP6A5 and CYP6A5v2, were isolated from a pyrethroid-resistant house fly stain, ALHF. The two alleles shared 98% similarity in amino acid sequence. To understand the importance of these two alleles in resistance and examine the expression profile of the two alleles between resistant and susceptible strains, quantitative real-time PCR (qRT-PCR) was performed and compared with the Northern blot analysis. We found that qRT-PCR was an efficient method to characterize the expression profiles between these two sequence-closely-related P450 genes between resistant and susceptible houses flies. One of them, CYP6A5v2, was constitutively overexpressed in ALHF house flies compared with susceptible house fly strains. Moreover, this gene was predominantly expressed in the abdominal tissues of ALHF, in which the primary detoxification organs of insects are located. However, there was no significant difference in the expression of CYP6A5 between ALHF and susceptible house flies. The genetic linkage analysis was conducted to determine the possible link between the constitutively overexpressed CYP6A5v2 and insecticide resistance. CYP6A5v2 was mapped on autosome 5, which is correlated with the linkage of resistance in ALHF. Taken together, the study suggests the importance of CYP6A5v2 in increasing metabolic detoxification of insecticides in ALHF. The distinct expression of CYP6A5 and CYP6A5v2 in resistant and susceptible house flies implies the functional difference of theses two genes in house flies and suggests that they are two recently diverged P450 genes presented in a single organism.     

Effect of spermine on the inhibition by fatty acid on AMP deaminase reaction as a control system of the adenylate energy charge in yeast

Treatment of rats with pyrazole elevated the hepatic microsomal dimethylnitrosamine demethylase activity (DMNd) by several fold. Methylethylnitrosamine demethylase activity was also increased by pyrazole, but some classical monooxygenase activities were not induced. The treatment induced a new protein species which has an apparent molecular weight of 52,000 dal and is believed to be a cytochrome P-450 isozyme. The involvement of a hemoprotein in the pyrazole-induced DMNd was demonstrated in an experiment with CoCl₂ which decreased both the microsomal cytochrome P-450 content and DMNd. The induced enzyme with a single K_m value of 0.061 mM and V_max of 12.1 nmol/min/mg is probably the most efficient enzyme known to metabolize nitrosamines. NADPH-cytochrome P-450 reductase was also demonstrated to be an essential component enzyme of the DMNd. These results further substantiate the idea that the P-450-containing monooxygenase is responsible for the metabolism of dimethylnitrosamine in both the control and pyrazole induced microsomes.     

Possible role for covalent modification in the reversible activation of ecdysone 20-monooxygenase activity

Evidence is presented for the reversible activation-inactivation of the microsomal ecdysone 20-monooxygenase from fat body of the cotton leafworm, Spodoptera littoralis, in a manner commensurate with reversible changes in its phosphorylation state. The activity of the monooxygenase was higher following preincubation with fluoride (an inhibitor of phosphoprotein phosphatases) than in its absence. Preincubation with alkaline phosphatase or with cAMP-dependent protein kinase resulted in appreciable diminution or enhancement, respectively, in monooxygenase activity. Activation of ecdysone 20-monooxygenase activity could also be effected by incubation with a cytosolic fraction in the presence of cAMP, ATP, and fluoride; this activation was prevented by a cAMP-dependent protein kinase inhibitor. Similarly, inactivation of the monooxygenase was achieved by preincubation with cytosol, the effect being enhanced by Ca²⁺-calmodulin or by Mg²⁺ ions. The combined results provide indirect evidence that the microsomal ecdysone 20-monooxygenase exists in an active phosphorylated form and an inactive dephosphorylated form, interconvertible by a cAMP-dependent protein kinase and a phosphoprotein phosphatase.     

Biochemical quantitation and histochemical localization of glucose-6-phosphate dehydrogenase activity in the olfactory system of adult and aged rats

The activity of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the hexose monophosphate shunt, was examined in olfactory epithelium, respiratory epithelium, olfactory bulb, and occipital cortex in Fisher 344 rats aged 4 and 24 months. Marked differences in this enzyme were found in olfactory compared to nonolfactory tissues. Olfactory epithelium and olfactory bulb have much greater glucose-6-phosphate dehydrogenase activity than respiratory epithelium and occipital cortex at both ages. Glucose-6-phosphate dehydrogenase remains fairly constant between adulthood and senescence in respiratory epithelium and occipital cortex. However, glucose-6-phosphate dehydrogenase activity decreases during the same time in both of the olfactory tissues examined. Previous studies of changes in this enzyme with aging have shown increases in enzyme activity in some brain regions, but never the decreases that we describe in olfactory tissues. Glucose-6-phosphate dehydrogenase histochemistry revealed intense staining of both the apical layer of olfactory epithelium and of Bowman's glands along with their ducts. Histochemistry of the olfactory bulb showed strongest staining in the nerve and glomerular layers of the bulb. The functional implications of these findings are discussed.